Nucleic acid labeling compounds

ABSTRACT

Nucleic acid labeling compounds containing heterocyclic derivatives are disclosed. The heterocyclic derivative containing compounds are synthesized by condensing a heterocyclic derivative with a cyclic group (e.g. a ribofuranose derivative). The labeling compounds are suitable for enzymatic attachment to a nucleic acid, either terminally or internally, to provide a mechanism of nucleic acid detection.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

[0001] This invention was made with Government support under contract70NANB5H1031 awarded by the Advanced Technology Program of the NationalInstitute of Standards and Technology. The Government has certain rightsin this invention.

TECHNICAL FIELD

[0002] The present invention relates to nucleic acid labeling compounds.More specifically, the invention provides heterocyclic derivativescontaining a detectable moiety. The invention also provides methods ofmaking such heterocyclic derivatives. It further provides methods ofattaching the heterocyclic derivatives to a nucleic acid.

BACKGROUND ART

[0003] Gene expression in diseased and healthy individuals is oftentimesdifferent and characterizable. The ability to monitor gene expression insuch cases provides medical professionals with a powerful diagnostictool. This form of diagnosis is especially important in the area ofoncology, where it is thought that the overexpression of an oncogene, orthe underexpression of a tumor suppressor gene, results intumorogenesis. See Mikkelson et al. J. Cell. Biochem. 1991, 46, 3-8.

[0004] One can indirectly monitor gene expression, for example, bymeasuring a nucleic acid (e.g., mRNA) that is the transcription productof a targeted gene. The nucleic acid is chemically or biochemicallylabeled with a detectable moiety and allowed to hybridize with alocalized nucleic acid probe of known sequence. The detection of alabeled nucleic acid at the probe position indicates that the targetedgene has been expressed. See International Application Publication Nos.WO 97/27317, WO 92/10588 and WO 97/10365.

[0005] The labeling of a nucleic acid is typically performed bycovalently attaching a detectable group (label) to either an internal orterminal position. Scientists have reported a number of detectablenucleotide analogues that have been enzymatically incorporated into anoligo- or polynucleotide. Langer et al., for example, disclosedanalogues of dUTP and UTP that contain a covalently bound biotin moiety.Proc. Natl. Acad. Sci. USA 1981, 78, 6633-6637. The analogues, shownbelow, possess an allylamine linker arm that is attached to the C-5position of the pyrimidine ring. The dUTP and UTP analogues, wherein Ris H or OH, were incorporated into a polynucleotide.

[0006] Petrie et al. disclosed a dATP analogue,3-[5-[(N-biotinyl-6-aminocaproyl)-amino]pentyl]-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine-5′-triphosphate.Bioconjugate Chem. 1991, 2, 441-446. The analogue, shown below, ismodified at the 3-position with a linker arm that is attached to abiotin moiety. Petrie et al. reported that the compound wherein R isbiotin is incorporated into DNA by nick translation.

[0007] Prober et al. disclosed a set of four dideoxynucleotides, eachcontaining a succinylfluorescein dye. Science 1987, 238, 336-341. Thedideoxynucleotides, one of which is shown below, were enzymaticallyincorporated into an oligonucleotide through a template directedextension of a primer. The compounds provided for a DNA sequencingmethod based on gel migration.

[0008] Herrein et al. disclosed modified nucleoside trisphosphates ofthe four DNA bases. Helv. Chim. Acta 1994, 77, 586-596. The compounds,one of which is shown below, contain a 3′-amino group containingradioactive or fluorescent moieties. Herrlein et al. further describedthe use of the nucleoside analogues as DNA chain terminators.

[0009] Cech et al. disclosed 3′-amino-functionalized nucleosidetriphosphates. Collect. Czech. Chem. Commun. 1996, 61, S297-S300. Thecompounds, one of which is shown below, contain a fluorescein attachedto the 3′-position through an amino linker. Cech et al. proposed thatthe described functionalized nucleosides would be useful as terminatorsfor DNA sequencing.

DISCLOSURE OF THE INVENTION

[0010] The development of a novel nucleic acid labeling compound that iseffectively incorporated into a nucleic acid to provide a readilydetectable composition would benefit genetic analysis technologies. Itwould aid, for example, in the monitoring of gene expression and thedetection and screening of mutations and polymorphisms. Such a compoundshould be suitable for enzymatic incorporation into a nucleic acid.Furthermore, the nucleic acid to which the labeling compound is attachedshould maintain its ability to bind to a probe, such as a complementarynucleic acid.

[0011] The present invention provides nucleic acid labeling compoundsthat are capable of being enzymatically incorporated into a nucleicacid. The nucleic acids to which the compounds are attached maintaintheir ability to bind to a complementary nucleic acid sequence.

[0012] The nucleic acid labeling compounds of the present invention areof the following structure:

A-O—CH₂-T-H_(c)-L-(M)_(m)-Q

[0013] wherein A is hydrogen or a finctional group that permits theattachment of the nucleic acid labeling compound to a nucleic acid; T isa template moiety; H_(c) is a heterocyclic group; L is a linker moiety;Q is a detectable moiety; and M is a connecting group, wherein m is aninteger ranging from 0 to about 5.

[0014] In one embodiment, the nucleic acid labeling compounds are of thefollowing structure:

[0015] wherein A is H or a functional group that permits the attachmentof the nucleic acid labeling compound to a nucleic acid; X is O, S, NR₁or CHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₁, SR₁ or NHR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, For OR₁, wherein R₁ is H, alkyl or aryl; L is amido alkyl; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about 3.

[0016] In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₁,wherein R₁ is H, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H,alkyl or aryl; L is —C(O)NH(CH₂)_(n)NH—, wherein n is an integer rangingfrom about 2 to about 1; Q is biotin or a carboxyfluorescein; and, M is—CO(CH₂)₅NH—, wherein m is 1 or 0.

[0017] In another embodiment, Y is H or OH; Z is H or OH; L is—C(O)NH(CH₂)₄NH—; Q is biotin; and, M is —CO(CH₂)₅NH, wherein m is 1.

[0018] In another embodiment, Y is H or OH; Z is H or OH; L is—C(O)NH(CH₂)₄NH—; Q is 5-carboxyfluorescein; and, m is 0.

[0019] In one embodiment, the nucleic acid labeling compounds are of thefollowing structure:

[0020] wherein A is H or a functional group that permits the attachmentof the nucleic acid labeling compound to a nucleic acid; X is O, S, NR₁or CHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₁, SR₁ or NHR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, For OR₁, wherein R₁ is H, alkyl or aryl; L is amino alkyl; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about 3.

[0021] In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₁,wherein R₁ is H, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H,alkyl or aryl; L is —NH(CH₂)_(n)NH—, wherein n is an integer rangingfrom about 2 to about 10; Q is biotin or carboxyfluorescein; and, M is—CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

[0022] In another embodiment, Y is H or OH; Z is H or OH; L is—NH(CH₂)₄NH—; Q is biotin; and, m is 0.

[0023] In another embodiment, Y is H or OH; Z is H or OH; L is—NH(CH₂)₄NH—; Q is 5-carboxyfluorescein; and, m is 0.

[0024] In one embodiment, the nucleic acid labeling compounds are of thefollowing structure:

[0025] wherein A is H or a functional group that permits the attachmentof the nucleic acid labeling compound to a nucleic acid; X is O, S, NR₁or CHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₁, SR₁ or NHR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, For OR₁, wherein R₁ is H, alkyl or aryl; L is alkynyl alkyl; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about 3.

[0026] In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₁,wherein R₁ is H, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H,alkyl or aryl; L is —C≡C(CH₂)_(n)NH—, wherein n is an integer rangingfrom about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is—CO(CH₂)₅NH—, wherein m is 1 or 0.

[0027] In another embodiment, Y is H or OH; Z is H or OH; L is—C≡CCH₂NH—; Q is biotin; and, m is 1.

[0028] In another embodiment, Y is H or OH; Z is H or OH; L is—C≡CCH₂NH—; Q is 5-carboxyfluorescein; and, m is 1.

[0029] In one embodiment, the nucleic acid labeling compounds are of thefollowing structure:

[0030] wherein A is H or a functional group that permits the attachmentof the nucleic acid labeling compound to a nucleic acid; X is O, S, NR₁or CHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₁, SR₁ or NHR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, For OR₁, wherein R₁ is H, alkyl or aryl; L is amino alkyl; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about 3.

[0031] In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₁,wherein R₁ is H, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H,alkyl or aryl; L is —NH(CH₂)_(n)NH—, wherein n is an integer rangingfrom about 2 to about 10; Q is biotin or carboxyfluorescein; and, M is—CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

[0032] In another embodiment, Y is H or OH; Z is H or OH; L is—NH(CH₂)₄NH—; Q is biotin; and, M is —CO(CH₂)₅NH—, wherein m is 1.

[0033] In another embodiment, Y is H or OH; Z is H or OH; L is—NH(CH₂)₄NH—; Q is 5-carboxyfluorescein; and, M is —CO(CH₂)₅NH—, whereinm is 1.

[0034] In one embodiment, the nucleic acid labeling compounds are of thefollowing structure:

[0035] wherein A is H or a functional group that permits the attachmentof the nucleic acid labeling compound to a nucleic acid; X is O, S, NR₁or CHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₁, SR₁ or NHR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, For OR₁, wherein R₁ is H, alkyl or aryl; L is functionalized alkyl,alkenyl alkyl or alkynyl alkyl; Q is a detectable moiety; and, M is aconnecting group, wherein m is an integer ranging from 0 to about 3.

[0036] In another embodiment, A is H or H₄ _(O) ₉P₃—; X is O; Y is H orOR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ isH, alkyl or aryl; L is —CH═CH(CH₂)_(n)NH—, wherein n is an integerranging from about 1 to about 10; Q is biotin or carboxyfluorescein;and, M is —CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

[0037] In another embodiment, Y is H or OH; Z is H or OH; L is—CH═CHCH₂NH—; Q is biotin; and, m is 0.

[0038] In another embodiment, Y is H or OH; Z is H or OH; L is—CH═CHCH₂NH—; Q is 5-carboxyfluorescein; and, m is 0.

[0039] In one embodiment, the nucleic acid labeling compounds are of thefollowing structure:

[0040] wherein A is H or a functional group that permits the attachmentof the nucleic acid labeling compound to a nucleic acid; X is O, S, NR₁or CHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₁, SR₁ or NHR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, For OR₁, wherein R₁ is H, alkyl or aryl; L is functionalized alkyl,alkenyl alkyl or alkynyl alkyl; Q is a detectable moiety; and, M is aconnecting group, wherein m is an integer ranging from 0 to about 3.

[0041] In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₁,wherein R₁ is H, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H,alkyl or aryl; L is —CH═CH(CH₂)_(n)NH—, wherein n is an integer rangingfrom about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is—CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

[0042] In another embodiment, Y is H or OH; Z is H or OH; L is—CH═CHCH₂NH—; Q is biotin; and, m is 0.

[0043] In another embodiment, Y is H or OH; Z is H or OH; L is—CH=CHCH₂NH—; Q is 5-carboxyfluorescein; and, m is 0.

[0044] The present invention also provides nucleic acid derivativesproduced by coupling a nucleic acid labeling compound with a nucleicacid and hybridization products comprising the nucleic acid derivativesbound to a complementary probe.

[0045] In one embodiment, the nucleic acid labeling compound used in thecoupling is of the following structure:

[0046] wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ isH, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl;L is —C(O)NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2to about 10; Q is biotin or a carboxyfluorescein; and, M is—CO(CH₂)₅NH—, wherein m is 1 or 0.

[0047] The hybridization product formed from this nucleic acidderivative comprises the nucleic acid derivative bound to acomplementary probe. In one embodiment, the probe is attached to a glasschip.

[0048] In another embodiment, the nucleic acid labeling compound used inthe coupling is of the following structure:

[0049] wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ isH, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl;L is —NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

[0050] The hybridization product formed from this nucleic acidderivative comprises the nucleic acid derivative bound to acomplementary probe. In one embodiment, the probe is attached to a glasschip.

[0051] In another embodiment, the nucleic acid labeling compound used inthe coupling is of the following structure:

[0052] wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ isH, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl;L is —C≡C(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH—,wherein m is 1 or 0.

[0053] The hybridization product formed from this nucleic acidderivative comprises the nucleic acid derivative bound to acomplementary probe. In one embodiment, the probe is attached to a glasschip.

[0054] In another embodiment, the nucleic acid labeling compound used inthe coupling is of the following structure:

[0055] wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ isH, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl;L is —NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

[0056] The hybridization product formed from this nucleic acidderivative comprises the nucleic acid derivative bound to acomplementary probe. In one embodiment, the probe is attached to a glasschip.

[0057] In another embodiment, the nucleic acid labeling compound used inthe coupling is of the following structure:

[0058] wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ isH, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl;L is —CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

[0059] The hybridization product formed from this nucleic acidderivative comprises the nucleic acid derivative bound to acomplementary probe. In one embodiment, the probe is attached to a glasschip.

[0060] In another embodiment, the nucleic acid labeling compound used inthe coupling is of the following structure:

[0061] wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ isH, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl;L is —CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

[0062] The hybridization product formed from this nucleic acidderivative comprises the nucleic acid derivative bound to acomplementary probe. In one embodiment, the probe is attached to a glasschip.

[0063] The present invention also provides methods of synthesizingnucleic acid derivatives by attaching a nucleic acid labeling compoundto a nucleic acid. It further provides methods of detecting nucleicacids involving incubating the nucleic acid derivatives with a probe.

[0064] In one embodiment, the nucleic acid labeling compound attached tothe nucleic acid is of the following structure:

[0065] wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ isH, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl;L is —C(O)NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2to about 10; Q is biotin or a carboxyfluorescein; and, M is—CO(CH₂)₅NH—, wherein m is 1 or 0.

[0066] The method of nucleic acid detection using the nucleic acidderivative involves the incubation of the derivative with a probe. Inone embodiment, the probe is attached to a glass chip.

[0067] In one embodiment, the nucleic acid labeling compound attached tothe nucleic acid is of the following structure:

[0068] wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ isH, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl;L is —NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

[0069] The method of nucleic acid detection using the nucleic acidderivative involves the incubation of the derivative with a probe. Inone embodiment, the probe is attached to a glass chip.

[0070] In one embodiment, the nucleic acid labeling compound attached tothe nucleic acid is of the following structure:

[0071] wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ isH, alkyl or aryl;

[0072] Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl; L is—C≡C(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 to about10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH—, wherein mis 1 or 0.

[0073] The method of nucleic acid detection using the nucleic acidderivative involves the incubation of the derivative with a probe. Inone embodiment, the probe is attached to a glass chip.

[0074] In one embodiment, the nucleic acid labeling compound attached tothe nucleic acid is of the following structure:

[0075] wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ isH, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl;L is —NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

[0076] The method of nucleic acid detection using the nucleic acidderivative involves the incubation of the derivative with a probe. Inone embodiment, the probe is attached to a glass chip.

[0077] In one embodiment, the nucleic acid labeling compound attached tothe nucleic acid is of the following structure:

[0078] wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ isH, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl;L is —CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

[0079] The method of nucleic acid detection using the nucleic acidderivative involves the incubation of the derivative with a probe. Inone embodiment, the probe is attached to a glass chip.

[0080] In one embodiment, the nucleic acid labeling compound attached tothe nucleic acid is of the following structure:

[0081] wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ isH, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl;L is —CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

[0082] The method of nucleic acid detection using the nucleic acidderivative involves the incubation of the derivative with a probe. Inone embodiment, the probe is attached to a glass chip.

BRIEF DESCRIPTION OF THE DRAWINGS

[0083]FIG. 1 shows a nonlimiting set of template moieties.

[0084]FIG. 2 shows a nonlimiting set of heterocyclic groups:4-arninopyrazolo[3,4-d]pyrimidine, pyrazolo[3,4-d]pyrimidine,1,3-diazole (imidazole), 1,2,4-triazine-3-one, 1,2,4-triazine-3,5-dioneand 5-amino-1,2,4-triazine-3-one.

[0085]FIG. 3 shows a synthetic route to fluorescein and biotin labeled1-(2,3-dideoxy-D-glycero-pentafuranosyl)imidazole-4-carboxamidenucleotides.

[0086]FIG. 4 shows a synthetic route to C3-labeled4-aminopyrazolo[3,4-d]pyrimidine β-D-ribofuranoside triphosphates.

[0087]FIG. 5 shows a synthetic route to fluorescein and biotin labeledN6-dideoxy-pyrazolo[3,4-d]pyrimidine nucleotides.

[0088]FIG. 6 shows a synthetic route to N4-labeled 1,2,4-triazine-3-oneβ -D-ribofuranoside triphosphates.

[0089]FIG. 7 shows a synthetic route to biotin and fluoresceinC5-labeled 1,2,4-triazine-3,5-dione riboside triphosphates.

[0090]FIG. 8 shows a synthetic route to biotin and fluoresceinC5-labeled 5-amino-1,2,4-triazine-3-one riboside triphosphates.

[0091]FIG. 9 shows graphical comparisons of observed hybridizationfluorescence intensities using Fluorescein-ddITP and Fluorescein-ddATP.

[0092]FIG. 10 shows a graphical comparison of observed hybridizationfluorescence intensities using Biotin-(M)₂-ddAPTP (whereinM=aminocaproyl) and Biotin-N6-ddATP.

[0093]FIG. 11 shows graphical comparisons of observed hybridizationfluorescence intensities using Biotin-M-ddITP (wherein M=aminocaproyl)and Biotin-N6-ddATP.

[0094]FIG. 12 shows a graphical comparison of overall re-sequencing(base-calling) accuracy using Fluorescein-ddITP and Fluorescein-N6-ddATPlabeled targets.

[0095]FIG. 13 shows a graphical comparison of overall re-sequencingaccuracy using Biotin-M-ddITP (wherein M=aminocaproyl) andBiotin-N6-ddATP.

[0096]FIG. 14 shows a graphical comparison of re-sequencing accuracyusing Biotin-(M)₂-ddAPPTP (wherein M=aminocaproyl) and Biotin-N6-ddATP.

BEST MODE FOR CARRYING OUT THE INVENTION

[0097] Definitions

[0098] “Alkyl” refers to a straight chain, branched or cyclic chemicalgroup containing only carbon and hydrogen. Alkyl groups include, withoutlimitation, ethyl, propyl, butyl, pentyl, cyclopentyl and 2-methylbutyl.Alkyl groups are unsubstituted or substituted with 1 or moresubstituents (e.g., halogen, alkoxy, amino).

[0099] “Aryl” refers to a monovalent, unsaturated aromatic carbocyclicgroup. Aryl groups include, without limitation, phenyl, naphthyl,anthryl and biphenyl. Aryl groups are unsubstituted or substituted with1 or more substituents (e.g. halogen, alkoxy, amino).

[0100] “Amido alkyl” refers to a chemical group having the structure—C(O)NR₃R₄—, wherein R₃ is hydrogen, alkyl or aryl, and R₄ is alkyl oraryl. Preferably, the amido alkyl group is of the structure—C(O)NH(CH₂)_(n)R₅—, wherein n is an integer ranging from about 2 toabout 10, and R₅ is O, NR_(6,) or C(O), and wherein R₆ is hydrogen,alkyl or aryl. More preferably, the amido alkyl group is of thestructure —C(O)NH(CH₂)_(n)N(H)—, wherein n is an integer ranging fromabout 2 to about 6. Most preferably, the amido alkyl group is of thestructure —C(O)NH(CH₂)₄N(H)—.

[0101] “Alkynyl alkyl” refers to a chemical group having the structure—C≡—C—R₄—, wherein R₄ is alkyl or aryl. Preferably, the alkynyl alkylgroup is of the structure —C≡C—(CH₂)_(n)R₅—, wherein n is an integerranging from 1 to about 10, and R₅ is O, NR₆ or C(O), wherein R₆ ishydrogen, alkyl or aryl. More preferably, the alkynyl alkyl group is ofthe structure —C≡C—(CH₂)_(n)N(H)—, wherein n is an integer ranging from1 to about 4. Most preferably, the alkynyl alkyl group is of thestructure —C≡C—CH₂N(H)—.

[0102] “Alkenyl alkyl” refers to a chemical group having the structure—CH═CH—R₄—, wherein R₄ is alkyl or aryl. Preferably, the alkenyl alkylgroup is of the structure —CH═CH—(CH₂)_(n)R₅—, wherein n is an integerranging from 1 to about 10, and R₅ is O, NR₆ or C(O), wherein R₆ ishydrogen, alkyl or aryl. More preferably, the alkenyl alkyl group is ofthe structure —CH═CH—(CH₂)_(n)N(H)—, wherein n is an integer rangingfrom 1 to about 4. Most preferably, the alkenyl alkyl group is of thestructure —CH═CH—CH₂N(H)—.

[0103] “Functionalized alkyl” refers to a chemical group of thestructure —(CH₂)_(n)R₇—, wherein n is an integer ranging from 1 to about10, and R₇ is O, S, NH or C(O). Preferably, the functionalized alkylgroup is of the structure —(CH₂)_(n)C(O)—, wherein n is an integerranging from 1 to about 4. More preferably, the functionalized alkylgroup is of the structure —CH₂C(O)—.

[0104] “Alkoxy” refers to a chemical group of the structure—O(CH₂)_(n)R₈—, wherein n is an integer ranging from 2 to about 10, andR₈ is O, S, NH or C(O). Preferably, the alkoxy group is of the structure—O(CH₂)_(n)C(O)—, wherein n is an integer ranging from 2 to about 4.More preferably, the alkoxy group is of the structure —OCH₂CH₂C(O)—.

[0105] “Thio” refers to a chemical group of the structure—S(CH₂)_(n)R₈—, wherein n is an integer ranging from 2 to about 10, andR₈ is O, S, NH or C(O). Preferably, the thio group is of the structure—S(CH₂)_(n)C(O)—, wherein n is an integer ranging from 2 to about 4.More preferably, the thio group is of the structure —SCH₂CH₂C(O)—.

[0106] “Amino alkyl” refers to a chemical group having an amino groupattached to an alkyl group. Preferably an amino alkyl is of thestructure —NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2to about 10. More preferably it is of the structure —NH(CH₂)_(n)NH—,wherein n is an integer ranging from about 2 to about 4. Mostpreferably, the amino alkyl group is of the structure —NH(CH₂)₄NH—.

[0107] “Nucleic acid” refers to a polymer comprising 2 or morenucleotides and includes single-, double- and triple stranded polymers.“Nucleotide” refers to both naturally occurring and non-naturallyoccurring compounds and comprises a heterocyclic base, a sugar, and alinking group, preferably a phosphate ester. For example, structuralgroups may be added to the ribosyl or deoxyribosyl unit of thenucleotide, such as a methyl or allyl group at the 2′-O position or afluoro group that substitutes for the 2′-O group. The linking group,such as a phosphodiester, of the nucleic acid may be substituted ormodified, for example with methyl phosphonates or O-methyl phosphates.Bases and sugars can also be modified, as is known in the art. “Nucleicacid,” for the purposes of this disclosure, also includes “peptidenucleic acids” in which native or modified nucleic acid bases areattached to a polyamide backbone.

[0108] “Probe” refers to a nucleic acid that can be used to detect, byhybridization, a target nucleic acid. Preferably, the probe iscomplementary to the target nucleic acid along the entire length of theprobe, but hybridization can occur in the presence of one or more basemismatches between probe and target.

[0109] Nucleic Acid Labeling Compounds

[0110] The nucleic acid labeling compounds of the present invention areof the following structure:

A-O—CH₂-T-H_(c)-L-(M)_(m)-Q

[0111] wherein A is hydrogen or a functional group that permits theattachment of the nucleic acid labeling compound to a nucleic acid; T isa template moiety; H_(c) is a heterocyclic group; L is a linker moiety;Q is a detectable moiety; and M is an connecting group, wherein m is aninteger ranging from 0 to about 5.

[0112] The group A is either hydrogen or a functional group that permitsthe attachment of a nucleic acid labeling compound to a nucleic acid.Nonlimiting examples of such groups include the following:monophosphate; diphosphate; triphosphate (H₄O₉P); phosphoramidite((R₂N)(R′O)P), wherein R is linear, branched or cyclic alkyl, and R′ isa protecting group such as 2-cyanoethyl; and H-phosphonate(HP(O)O—HNR₃), wherein R is linear, branched or cyclic alkyl.

[0113] The template moiety (T) is covalently attached to a methylenegroup (CH₂) at one position and a heterocyclic group (H_(c)) at anotherposition. A nonlimiting set of template moieties is shown in FIG. 1,wherein the substituents are defined as follows: X is O, S, NR₁ or CHR₂;Y is H, N₃, F, OR₁, SR₁ or NHR₁; Z is H, N₃, F or OR₁; W is O, S or CH₂;D is O or S; and, G is O, NH or CH₂. The substituents R₁ and R₂ areindependent of one another and are H, alkyl or aryl.

[0114] The heterocyclic group (H_(c)) is a cyclic moiety containing bothcarbon and a heteroatom. Nonlimiting examples of heterocyclic groupscontemplated by the present invention are shown in FIG. 2.:4-aminopyrazolo[3,4-d]pyrimidine; pyrazolo[3,4-d]pyrimidine; 1,3-diazole(imidazole); 1,2,4-triazine-3-one; 1,2,4-triazine-3,5-dione; and,5-amino-1,2,4-triazine-3 -one.

[0115] The linker moiety (L) of the nucleic acid labeling compound iscovalently bound to the heterocycle (H_(c)) at one terminal position. Itis attached to the detectable moiety (Q) at another terminal position,either directly or through a connecting group (M). It is of a structurethat is sterically and electronically suitable for incorporation into anucleic acid. Nonlimiting examples of linker moieties include amidoalkyl groups, alkynyl alkyl groups, alkenyl alkyl groups, functionalizedalkyl groups, alkoxyl groups, thio groups and amino alkyl groups.

[0116] Amido alkyl groups are of the structure —C(O)NR₃R₄—, wherein R₃is hydrogen, alkyl or aryl, and R₄ is alkyl or aryl. The amido alkylgroup is preferably of the structure —C(O)NH(CH₂)_(n)R₅—, wherein n isan integer ranging from about 2 to about 10 and R₅ is O, NR₆ or C(O),and wherein R₆ is hydrogen, alkyl or aryl. More preferably, the amidoalkyl group is of the structure —C(O)NH(CH₂)_(n)N(H)—, wherein n is aninteger ranging from about 2 to about 6. Most preferably, the amidoalkyl group is of the structure —C(O)NH(CH₂)₄N(H)—.

[0117] Alkynyl alkyl groups are of the structure —C≡C—R₄—, wherein R₄ isalkyl or aryl. The alkynyl alkyl group is preferably of the structure—C≡C(CH₂)_(n)R₅—, wherein n is an integer ranging from 1 to about 10 andR₅ is O, NR₆ or C(O), and wherein R₆ is hydrogen, alkyl or aryl. Morepreferably, the alkynyl alkyl group is of the structure—C≡C—(CH₂)_(n)N(H)—, wherein n is an integer ranging from 1 to about 4.Most preferably, the alkynyl alkyl group is of the structure—C≡C—CH₂N(H)—.

[0118] Alkenyl alkyl groups are of the structure —CH═CH—R₄—, wherein R₄is alkyl or aryl. The alkenyl alkyl group is preferably of the structure—CH═CH(CH₂)R₅—, wherein n is an integer ranging from 1 to about 10, andR₅ is O, NR₆ or C(O), and wherein R₆ is hydrogen, alkyl or aryl. Morepreferably, the alkenyl alkyl group is of the structure—CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging from 1 to about 4.Most preferably, the alkenyl alkyl group is of the structure—CH═CHCH₂NH—.

[0119] Functionalized alkyl groups are of the structure —(CH₂)_(n)R₇—,wherein n is an integer ranging from 1 to about 10, and R₇ is O, S, NH,or C(O). The functionalized alkyl group is preferably of the structure—(CH₂)_(n)C(O)—, wherein n is an integer ranging from 1 to about 4. Morepreferably, the functionalized alkyl group is —CH₂C(O)—.

[0120] Alkoxy groups are of the structure —O(CH₂)_(n)R₈—, wherein n isan integer ranging from 2 to about 10, and R₈ is O, S, NH, or C(O). Thealkoxy group is preferably of the structure —O(CH₂)_(n)C(O)—, wherein nis an integer ranging from 2 to about 4. More preferably, the alkoxygroup is of the structure —OCH₂CH₂C(O)—.

[0121] Thio groups are of the structure —S(CH₂)_(n)R₈—, wherein n is aninteger ranging from 2 to about 10, and R₈ is O, S, NH, or C(O). Thethio group is preferably of the structure —S(CH₂)_(n)C(O)—, wherein n isan integer ranging from 2 to about 4. More preferably, the thio group isof the structure —SCH₂CH₂C(O)—.

[0122] Amino alkyl groups comprise an amino group attached to an alkylgroup. Preferably, amino alkyl groups are of the structure—NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 to about10. The amino alkyl group is more preferably of the structure—NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 to about4. Most preferably, the amino alkyl group is of the structure—NH(CH₂)₄NH—.

[0123] The detectable moiety (Q) is a chemical group that provides ansignal. The signal is detectable by any suitable means, includingspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. In certain cases, the signal is detectable by2 or more means.

[0124] The detectable moiety provides the signal either directly orindirectly. A direct signal is produced where the labeling groupspontaneously emits a signal, or generates a signal upon theintroduction of a suitable stimulus. Radiolabels, such as ³H, ¹²⁵I, ³⁵S,¹⁴C or ³²P, and magnetic particles, such as Dynabeads™, are nonlimitingexamples of groups that directly and spontaneously provide a signal.Labeling groups that directly provide a signal in the presence of astimulus include the following nonlimiting examples: colloidal gold(40-80 nm diameter), which scatters green light with high efficiency;fluorescent labels, such as fluorescein, texas red, rhodamine, and greenfluorescent protein (Molecular Probes, Eugene, Oregon), which absorb andsubsequently emit light; chemiluminescent or bioluminescent labels, suchas luminol, lophine, acridine salts and luciferins, which areelectronically excited as the result of a chemical or biologicalreaction and subsequently emit light; spin labels, such as vanadium,copper, iron, manganese and nitroxide free radicals, which are detectedby electron spin resonance (ESR) spectroscopy; dyes, such as quinolinedyes, triarylmethane dyes and acridine dyes, which absorb specificwavelengths of light; and colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. See U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241.

[0125] A detectable moiety provides an indirect signal where itinteracts with a second compound that spontaneously emits a signal, orgenerates a signal upon the introduction of a suitable stimulus. Biotin,for example, produces a signal by forming a conjugate with streptavidin,which is then detected. See Hybridization With Nucleic Acid Probes. InLaboratory Techniques in Biochemistry and Molecular Biology; Tijssen,P., Ed.; Elsevier: New York, 1993; Vol. 24. An enzyme, such ashorseradish peroxidase or alkaline phosphatase, that is attached to anantibody in a label-antibody-antibody as in an ELISA assay, alsoproduces an indirect signal.

[0126] A preferred detectable moiety is a fluorescent group. Flourescentgroups typically produce a high signal to noise ratio, thereby providingincreased resolution and sensitivity in a detection procedure.Preferably, the fluorescent group absorbs light with a wavelength aboveabout 300 nm, more preferably above about 350 nm, and most preferablyabove about 400 nm. The wavelength of the light emitted by thefluorescent group is preferably above about 310 nm, more preferablyabove about 360 nm, and most preferably above about 410 nm.

[0127] The fluorescent detectable moiety is selected from a variety ofstructural classes, including the following nonlimiting examples: 1- and2-aminonaphthalene, p,p′-diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p′-diaminobenzophenone imines,anthracenes, oxacarbocyanine, marocyanine, 3-aminoequilenin, perylene,bisbenzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,bis-3-aminopridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolyl phenylamine, 2-oxo-3-chromen, indole, xanthen,7-hydroxycoumarin, phenoxazine, salicylate, strophanthidin, porphyrins,triarylmethanes, flavin, xanthene dyes (e.g., fluorescein and rhodaminedyes); cyanine dyes; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes andfluorescent proteins (e.g., green fluorescent protein,phycobiliprotein).

[0128] A number of fluorescent compounds are suitable for incorporationinto the present invention. Nonlimiting examples of such compoundsinclude the following: dansyl chloride; fluoresceins, such as3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate; N-phenyl-l-amino-8-sulfonatonaphthalene; N-phenyl-2-amino-6-sulfonatonaphthanlene;4-acetamido-4-isothiocyanatostilbene-2,2′-disulfonic acid;pyrene-3-sulfonic acid; 2-toluidinonapththalene-6-sulfonate; N-phenyl,N-methyl 2-aminonaphthalene-6-sulfonate; ethidium bromide; stebrine;auromine-0,2-(9′-anthroyl)palmitate; dansyl phosphatidylethanolamin;N,N′-dioctadecyl oxacarbocycanine; N,N′ -dihexyl oxacarbocyanine;merocyanine, 4-(3′-pyrenyl)butryate; d-3-aminodesoxy-equilenin;12-(9′-anthroyl)stearate; 2-methylanthracene; 9-vinylanthracene;2,2′-(vinylene-p-phenylene)bisbenzoxazole; p-bis[2-(4-methyl-5-phenyloxazolyl)]benzene; 6-dimethylamino-1,2-benzophenzin; retinol; bis(3′-aminopyridinium)-1,10-decandiyl diiodide; sulfonaphthylhydrazone ofhellibrienin; chlorotetracycline;N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide;N-[p-(2-benzimidazolyl)phenyl]maleimide; N-(4-fluoranthyl)maleimide;bis(homovanillic acid); resazarin;4-chloro-7-nitro-2,1,3-benzooxadizole; merocyanine 540; resorufin; rosebengal and 2,4-diphenyl-3(2H)-furanone. Preferably, the fluorescentdetectable moiety is a fluorescein or rhodamine dye.

[0129] Another preferred detectable moiety is colloidal gold. Thecolloidal gold particle is typically 40 to 80 nm in diameter. Thecolloidal gold may be attached to a labeling compound in a variety ofways. In one embodiment, the linker moiety of the nucleic acid labelingcompound terminates in a thiol group (—SH), and the thiol group isdirectly bound to colloidal gold through a dative bond. See Mirkin etal. Nature 1996, 382, 607-609. In another embodiment, it is attachedindirectly, for instance through the interaction between colloidal goldconjugates of antibiotin and a biotinylated labeling compound. Thedetection of the gold labeled compound may be enhanced through the useof a silver enhancement method. See Danscher et al. J. Histotech 1993,16, 201-207.

[0130] The connecting group (M)_(m) may serve to covalently attach thelinker group (L) to the detectable moiety (Q). It is of any suitablestructure that will not interfere with the function of the labelingcompound. Nonlimiting examples of M groups include the following:—CO(CH₂)₅NH—, —CO—, —CO(O)—, —CO(NH)—, and —CO(CH₂)₅NHCO(CH₂)₅NH—;wherein, m is an integer ranging from 0 to about 5, preferably 0 toabout 3.

[0131] In one embodiment, the nucleic acid labeling compounds of thepresent invention are of the following structure:

[0132] wherein L is a linker moiety; Q is a detectable moiety; X is O,S, NR₁ or CHR₂; Y is H, N₃, F, OR₁, SR₁ or NHR₁; Z is H, N₃, F or OR₁;H_(c) is a heterocyclic group; A is H or a functional group that permitsthe attachment of the nucleic acid label to a nucleic acid; and, M is aconnecting group, wherein m is an integer ranging from 0 to about 3. Thesubstituents R₁ and R₂ are independent of one another and are H, alkylor aryl.

[0133] In one embodiment, the heterocyclic group (H_(c)) is animidazole, and the nucleic acid labeling compounds are of the followingstructure:

[0134] wherein L is a linker moiety; Q is a detectable moiety; X is O,S, NR₁ or CHR₂; Y is H, N₃, F, OR₁, SR₁ or NHR₁; Z is H, N₃, F or OR₁; Ais H or a functional group that permits the attachment of the nucleicacid label to a nucleic acid; and, M is a connecting group, wherein m isan integer ranging from 0 to about 3. The substituents R₁ and R₂ areindependent of one another and are H, alkyl or aryl.

[0135] In a preferred embodiment, the heterocyclic group (H_(c)) is animidazole and the linking moiety is amido alkyl:

[0136] wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; R₃is hydrogen or alkyl; R₄ is —(CH₂)_(n)NH—, wherein n is an integerranging from about 2 to about 10; Q is biotin or carboxyfluorescein; Ais hydrogen or H₄O₉P₃—; and, M is —CO(CH₂)₅NH— or —CO—, wherein m is 1or 0. More preferably, Y and Z are hydrogen; R₃ is hydrogen; R₄ is—(CH₂)₄NH—; A is H₄O₉P₃—; and, Q is biotin, wherein M is —CO(CH₂)₅NH—and m is 1, or 5- or 6-carboxyfluorescein, wherein m is 0.

[0137] In another embodiment, the heterocyclic group (H_(c)) is a C3substituted 4-amino-pyrazolo[3,4-d]pyrimidine, and the nucleic acidlabeling compounds are of the following structure:

[0138] wherein L is a linker moiety; Q is a detectable moiety; X is O,S, NR₁ or CHR₂; Y is H, N₃, F, OR₁, SR₁ or NHR₁; Z is H, N₃, F or OR₁; Ais H or a functional group that permits the attachment of the nucleicacid label to a nucleic acid; and, M is a connecting group, wherein m isan integer ranging from 0 to about 3. The substituents R₁ and R₂ areindependent of one another and are H, alkyl or aryl.

[0139] In a preferred embodiment, the heterocyclic group (H_(c)) is a C3substituted 4-aminopyrazolo[3,4-d]pyrimidine and the linking group is analkynyl alkyl:

[0140] wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; nis an integer ranging from 1 to about 10; R₅ is O or NH; A is hydrogenor H₄O₉P₃—; Q is biotin or carboxyfluorescein; M is —CO(CH₂)₅NH—,wherein m is 1 or 0. More preferably, Y and Z are OH; n is 1; R₅ is NH;A is H₄O₉P₃—; and, Q is biotin or 5-or 6-carboxyfluorescein, wherein mis 1.

[0141] In another embodiment, the heterocyclic group (H_(c)) is an C4substituted pyrazolo[3,4-d]pyrimidine, and the nucleic acid labelingcompounds are of the following structure:

[0142] wherein L is a linker moiety; Q is a detectable moiety; X is O,S, NR₁ or CHR₂; Y is H, N₃, F, OR₁, SR₁ or NHR₁; Z is H, N₃, F or OR₁; Ais H or a functional group that permits the attachment of the nucleicacid label to a nucleic acid; and, M is a connecting group, wherein m isan integer ranging from 0 to about 3. The substituents R₁ and R₂ areindependent of one another and are H, alkyl or aryl.

[0143] In a preferred embodiment, the heterocyclic group (H_(c)) is anN4 substituted 4-amino-pyrazolo[3,4-d]pyrimidine and the linking groupis an amino alkyl:

[0144] wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; nis an integer ranging from about 2 to about 10; A is hydrogen orH₄O₉P₃—; Q is biotin or carboxyfluorescein; M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0. More preferably, Y and Zare hydrogen; n is 4; A is H₄O₉P₃—; Q is biotin or 5- or6-carboxyfluorescein, wherein m is 0.

[0145] In another embodiment, the heterocyclic group (H_(c)) is a1,2,4-triazine-3-one, and the nucleic acid labeling compounds are of thefollowing structure:

[0146] wherein L is a linker moiety; Q is a detectable moiety; X is O,S, NR₁ or CHR₂; Y is H, N₃, F, OR₁, SR₁ or NHR₁; Z is H, N₃, F or OR₁; Ais H or a functional group that permits the attachment of the nucleicacid label to a nucleic acid; and, M is a connecting group, wherein m isan integer ranging from 0 to about 3. The substituents R₁ and R₂ areindependent of one another and are H, alkyl or aryl.

[0147] In a preferred embodiment, the heterocyclic group (H_(c)) is a1,2,4-triazine-3-one and the linking group is amino alkyl:

[0148] wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; nis an integer ranging from about 2 to about 10; A is hydrogen orH₄O₉P₃—; Q is biotin or carboxyfluorescein; M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0. More preferably, Y and Zare hydroxyl; n is 4; A is H₄O₉P₃—; Q is biotin or 5- or6-carboxyfluorescein, wherein M is —CO(CH₂)₅NH—, and m is 1.

[0149] In another embodiment, the heterocyclic group (H_(c)) is a1,2,4-triazine-3,5-dione, and the nucleic acid labeling compounds are ofthe following structure:

[0150] wherein L is a linker moiety; Q is a detectable moiety; X is O,S, NR₁ or CHR₂; Y is H, N₃, F, OR₁, SR₁ or NHR₁; Z is H, N₃, F or OR₁; Ais H or a functional group that permits the attachment of the nucleicacid label to a nucleic acid; and, M is a connecting group, wherein m isan integer ranging from 0 to about 3. The substituents R₁ and R₂ areindependent of one another and are H, alkyl or aryl.

[0151] In a preferred embodiment, the heterocyclic group (H_(c)) is a1,2,4-triazine-3,5-dione and the linking group is alkenyl alkyl:

[0152] wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; nis an integer ranging from about 1 to about 10; R₅ is NR₆, wherein R₆ ishydrogen, alkyl or aryl; A is hydrogen or H₄O₉P₃—; Q is biotin orcarboxyfluorescein; M is —CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—, whereinm is 1 or 0.

[0153] In another embodiment, the heterocyclic group (H_(c)) is a5-amino-1,2,4-triazine-3-one, and the nucleic acid labeling compoundsare of the following structure:

[0154] wherein L is a linker moiety; Q is a detectable moiety; X is O,S, NR₁ or CHR₂; Y is H, N₃, F, OR₁, SR₁ or NHR₁; Z is H, N₃, F or OR₁; Ais H or a functional group that permits the attachment of the nucleicacid label to a nucleic acid; and, M is a connecting group, wherein m isan integer ranging from 0 to about 3. The substituents R₁ and R₂ areindependent of one another and are H, alkyl or aryl.

[0155] In a preferred embodiment, the heterocyclic group (H_(c)) is a5-amino-1,2,4-triazine-3-one and the linking group is alkenyl alkyl:

[0156] wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; nis an integer ranging from about 1 to about 10; R₅ is NR₆, wherein R₆ ishydrogen, alkyl or aryl; A is hydrogen or H₄O₉P₃—; Q is biotin orcarboxyfluorescein; M is —CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—, whereinm is 1 or 0.

[0157] Synthesis of Nucleic Acid Labeling Compounds

[0158]FIG. 3 shows a synthetic route to nucleic acid labeling compounds8 a and 8 b, in which the heterocyclic group (H_(c)) is an imidazole andthe linker moiety (L) is an amido alkyl. The silyl protected imidazole(2) was added to pentofuranose (1) to provide a mixture ofcarboethoxyimidazole dideoxyriboside isomers (3 a-3 d). The isomers wereseparated to afford purified 3 c. The carboethoxy group of 3 c wasconverted into an amino carboxamide (4) upon treatment with a diamine.The terminal amine of 4 was protected to give the trifluoroacetylatedproduct 5. The silyl protecting group of 5 was removed, providing theprimary alcohol 6. Compound 6 was converted into a 5′ -triphosphate toafford 7. The trifluoroacetyl protecting group of 7 was removed, and thedeprotected amine was reacted with biotin-NH(CH₂)₅CO-NHS or5-carboxyfluorescein-NHS giving, respectively, nucleic acid labelingcompounds 8 a and 8 b.

[0159]FIG. 4 shows a synthetic route to C3-labeled4-aminopyrazolo[3,4-d]pyrimidine β-D-ribofuranoside triphosphates. Aprotected propargylamine linker was added to nucleoside (9) underpalladium catalysis to provide the coupled product (10). The primaryalcohol of the alkyne substituted nucleoside (10) was phosphorylated,yielding the 5′-triphosphate 11. The protected amine of triphosphate 11was then deprotected, and the resulting primary amine was treated with areactive biotin or fluorescein derivative to afford, respectively,nucleic acid labeling compounds 12 a and 12 b.

[0160]FIG. 5 shows a synthetic route to pyrazolopyrimidine nucleotides.A chloropyrazolopyrimidine (13) was added to pentofuranose 1 to provideadduct 14 as a mixture of anomers. A diamine was added to compound 14,affording a mixture of primary amines (15). The primary amines (15) wereprotected and chromatographically separated to yield the pure β-anomer16. The silyl group of 16 was removed and the resulting primary alcoholwas phosphorylated to provide triphosphate 17. The trifluoroacetyl groupof 17 was removed and the deprotected amine was treated with a reactivebiotin or carboxyfluorescein derivative giving, respectively, nucleicacid labeling compounds 18 a-18 d.

[0161]FIG. 6 shows a synthetic route to N4-labeled 1,2,4-triazine-3-oneβ -D-ribofuranoside triphosphates. 1,2,4-Triazine-3,5-dioneribonucleoside 19 was converted into the triazole nucleoside 20 upontreatment with triazole and phosphorous trichloride. Addition of adiamine to 20 provided aminoalkyl nucleoside 21. The primary amine of 21was protected, affording trifluoroacetamide 22. The primary alcohol of22 was phosphorylated, and the protected amine was deprotected andreacted with a reactive biotin or carboxyfluorescein derivative, giving,respectively, nucleic acid labeling compounds 23 a and 23 b.

[0162]FIG. 7 shows a synthetic route to C5-labeled1,2,4-triazine-3,5-dione riboside phosphates. Aldehyde 24 is reactedwith ylide 25 to provide the phthalimide protected allylamine 26.Compound 26 is coupled with pentofuranoside 27, yielding nucleoside 28.The phthalimide group of 28 is removed upon treatment with hydrazine toafford primary amine 29. Amine 29 is protected as amide 30. Amide 30 isphosphorylated, deprotected and treated with a reactive derivative ofbiotin or carboxyfluorescein, giving, respectively, nucleic acidlabeling compounds 31 a and 31 b.

[0163]FIG. 8 shows a synthetic route to C5-labeled5-amino-1,2,4-triazine-3-one riboside triphosphates. Compound 28 isconverted into the amino- 1,3-6-triazine compound 32 upon treatment witha chlorinating agent and ammonia. The phthalimide group of 32 is removedupon treatment with hydrazine, and the resulting primary amine isprotected to provide 33. Compound 33 is phosphorylated, deprotected andtreated with a reactive derivative of biotin or carboxyfluorescein,giving, respectively, nucleic acid labeling compounds 34 a and 34b.

[0164] Nucleic Acid Labeling

[0165] Nucleic acids can be isolated from a biological sample orsynthesized, on a solid support or in solution for example, according tomethods known to those of skill in the art. As used herein, there is nolimitation on the length or source of the nucleic acid used in alabeling process. Exemplary methods of nucleic acid isolation andpurification are described in Theory and Nucleic Acid Preparation. InLaboratory Techniques in Biochemistry and Molecular Biology:Hybridization With Nucleic Acid Probes; P. Tijssen, Ed.; Part I;Elsevier: N.Y., 1993. A preferred method of isolation involves an acidguanidinium-phenol-chloroform extraction followed by oligo dT columnchromotography or (dT)n magnetic bead use. Sambrook et al. MolecularCloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory,1989; Vols. 1-3; and Current Protocols in Molecular Biology; F. Ausubelet al. Eds.; Greene Publishing and Wiley Interscience: N.Y., 1987.

[0166] In certain cases, the nucleic acids are increased in quantitythrough amplification. Suitable amplification methods include, but arenot limited to, the following examples: polymerase chain reaction (PCR)(Innis, et al. PCR Protocols. A guide to Methods and Application;Academic Press: San Diego, 1990); ligase chain reaction (LCR) (Wu andWallace. Genomics 1989, 4, 560; Landgren, et al. Science 1988, 241,1077; and Barringer, et al. Gene 1990, 89, 117); transcriptionamplification (Kwoh et al. Proc. Natl. Acad. Sci. USA 1989, 86, 1173);and self-sustained sequence replication (Guatelli, et al. Proc. Nat.Acad. Sci. USA 1990, 87, 1874).

[0167] The nucleic acid labeling compound can be incorporated into anucleic acid using a number of methods. For example, it can be directlyattached to an original nucleic acid sample (e.g., MRNA, polyA mRNA,cDNA) or to an amplification product. Methods of attaching a labelingcompound to a nucleic acid include, without limitation, nicktranslation, 3-end-labeling, ligation, in vitro transcription (IVT) orrandom priming. Where the nucleic acid is an RNA, a labeledriboligonucleotide is ligated, for example, using an RNA ligase suchasT4 RNA Ligase. In The Enzymes; Uhlenbeck and Greensport, Eds.; Vol.XV, Part B, pp. 31-58; and, Sambrook et al., pp. 5.66-5.69. Terminaltransferase is used to add deoxy-, dideoxy- or ribonucleosidetriphosphates (dNTPs, ddNTPs or NTPs), for example, where the nucleicacid is single stranded DNA.

[0168] The labeling compound can also be incorporated at an internalposition of a nucleic acid. For example, PCR in the presence of alabeling compound provides an internally labeled amplification product.See, e.g., Yu et al. Nucleic Acids Research 1994, 22, 3226-3232.Similarly, IVT in the presence of a labeling compound can provide aninternally labeled nucleic acid.

[0169] Probe Hybridization

[0170] The nucleic acid to which the labeling compound is attached canbe detected after hybridization with a nucleic acid probe.Alternatively, the probe can be labeled, depending upon the experimentalscheme preferred by the user. The probe is a nucleic acid, or a modifiednucleic acid, that is either attached to a solid support or is insolution. It is complementary in structure to the labeled nucleic acidwith which it hybridizes. The solid support is of any suitable material,including polystyrene based beads and glass chips. In a preferredembodiment, the probe or target nucleic acid is attached to a glasschip, such as a GeneChip® product (Affymetrix, Inc., Santa Clara,Calif.). See International Publication Nos. WO 97/10365, WO 97/29212, WO97/27317, WO 95/11995, WO 90/15070, and U.S. Pat. Nos. 5,744,305 and5,445,934 which are hereby incorporated by reference.

[0171] Because probe hybridization is often a step in the detection of anucleic acid, the nucleic acid labeling compound must be of a structurethat does not substantially interfere with that process. The steric andelectronic nature of the labeling compound, therefore, is compatiblewith the binding of the attached nucleic acid to a complementarystructure.

EXAMPLES

[0172] The following examples are offered to illustrate, but not tolimit, the present invention.

[0173] General Experimental Details

[0174] Reagents were purchased from Aldrich Chemical Company (Milwaukee,Wis.) in the highest available purity. All listed solvents wereanhydrous. Intermediates were characterized by ¹H NMR and massspectrometry.

Example 1

[0175] Synthesis ofFluorescein- and Biotin-labeled 1-(2,3-dideoxy-β-D-glycero-pentafuranosyl)imidazole-4-carboxamide nucleotides.

[0176]1-O-acetyl-5-O-(t-butyldimethylsilyl)-2,3-dideoxy-D-glycero-pentafuranose1 (9.4 g, 34.2 mmole) (see, Duelholm, K.; Penderson, E. B., Synthesis,1992, 1) and 1-trimethylsilyl-4-carboethoxyimidazole 2 (6.3 g; 34.2mmole) (see, Pochet, S, et. al., Bioorg. Med. Chem. Lett., 1995, 5,1679) were combined in 100 ml dry DCM under Ar, and trimethylsilyltriflate catalyst (6.2 ml; 34.2 mmole) was added at 0° C. The solutionwas allowed to stir at room temperature for 5 hours and was then washed3× with 100 ml of saturated aqueous NaHCO₃, 1× with saturated aqueousNaCl, dried with NaSO₄ and evaporated to provide 14 g of a crude mixtureof four carboethoxyimidazole dideoxyriboside isomers (3 a-d),corresponding to α and β-anomers of both N1 and N3 alkylation products.The isomeric products were purified and separated by flashchromatography (silica gel, EtOAc-hexane), in 52% total yield. The β-N1isomer (2.2 g; 18% yield), was identified by ¹H-NMR chemical shift andNOE data (see, Pochet, S, et. al., Bioorg. Med. Chem. Lett., 1995, 5,1679). Purified 3 c (0.5 g; 1.4 mmole) was heated with a 20-fold excessof 1,4-diaminobutane (3.0 ml, 30 mmole) neat at 145° C. for 4 hours, andthen the resulting mixture was diluted with 50 ml EtOAc, washed 3× withwater, 1× with brine, and dried with NaSO₄ and evaporated to provide 500mg (95%) of the imidazole-4-(4-aminobutyl)carboxamide dideoxyriboside 4as a colorless oil. After coevaporation with toluene, 4 (393 mg; 0.75mmole) was combined with trifluoroacetylimidazole (94 uL; 0.83 mmole) in5 ml dry THF at 0° C., and stirred for 10 minutes. The solvent wasevaporated, and the oily residue taken up in 50 ml EtOAc, extracted 2×with saturated aqueous NaHCO₃, 1× with saturated aqueous NaCl, driedwith NaSO₄, and evaporated to yield 475 mg (99%) of the N-TFA protectednucleoside 5 as a colorless oil. The TBDMS group was removed by additionof excess triethylamine trihydrofluoride (2.3 ml; 14.4 mmole) in 20 mldry THF and stirring overnight. The THF was evaporated in vacuo, theresidue was taken up in 50 ml EtOAc and the solution was washedcarefully with a 1:1 mixture of saturated aqueous NaHCO₃ and brine untilneutral, then dried with NaSO₄, and evaporated to yield 340 mg (96%) ofthe 5 as a pale yellow oil. The NMR & MS data were consistent with theassigned structure.

[0177] Nucleoside 6 was converted to a 5′-triphosphate, deprotected,reacted with biotin-NH(CH₂)₅CO—NHS or 5-carboxyfluorescein-NHS andpurified according to procedures reported elsewhere (see, Prober, J. M.,et al., 1988, PCT 0 252 683 A2) to give the labeled nucleotides 8 a,bin >95% purity by HPLC, ³¹P-NMR.

Example 2

[0178] Synthesis of C3-Labeled 4-aminopyrazolo[3,4-d]pyrimidine β-D-ribofuranoside triphosphates.

[0179] The synthesis of 3-iodo-4-aminopyrazolo[3,4-d]pyrimidineribofuranside (9) was carried out as described by H. B. Cottam, et al.1993, J. Med. Chem. 36:3424. Using the appropriate deoxyfuranosideprecursors, both the 2′-deoxy and 2′,3′-dideoxy nucleosides are preparedusing analogous procedures. See, e.g., U. Neidballa & H. Vorbruggen1974, J. Org. Chem. 39:3654; K. L. Duehom & E. B. Pederson 1992,Synthesis 1992:1). Alternatively, these are prepared by deoxygenation ofribofuranoside 9 according to established procedures. See, M. J. Robinset al. 1983 J. Am. Chem. Soc. 103:4059; and, C. K. Chu, et al. 1989 J.Org. Chem. 54:2217.

[0180] A protected propargylamine linker was added to the4-aminopyrazolo[3,4-d] pyrimidine nucleoside (9) viaorganopalladium-mediated substitution to the 3-position of4-aminopyrazolo[3,4-d]pyrimidine riboside using the procedure describedby Hobbs (J. Org. Chem. 54: 3420; Science 238: 336.). Copper iodide (38mg; 0.2 mmole), triethylamine (560 uL; 4.0 mmole),N-trifluoroacetyl-3-aminopropyne (700 uL; 6.0 mmole) and3-iodo-4-aminopyrazolo[3,4-d]pyrimidine □-D-ribofuranoside (9) (H. B.Cottam, et al., 1993, J. Med. Chem. 36: 3424.) (786 mg; 2.0 mmole) werecombined in 5 ml of dry DMF under argon. To the stirring mixture wasadded tetrakis(triphenylphosphine) palladium(0) (232 mg; 0.2 mmole). Thesolution became homogeneous within 10 minutes, and was left stirring foran additional 4 hours in the dark, at which time the reaction wasdiluted with 20 mL of MeOH-DCM (1:1), 3.3 g of Dowex AG-1 anion exchangeresin (bicarbonate form) was added, and stirring was continued foranother 15 minutes. The resin was removed by filtration and washed withMeOH-DCM (1:1), and the combined filtrates were evaporated to dryness.The residue was dissolved in 4 mL of hot MeOH, then 15 mL DCM was addedand the mixture kept warm to maintain a homogeneous solution while itwas loaded onto a 5 cm×25 cm column of silica gel that had been packedin 1:9 MeOH-DCM. The product (R_(f)˜0.4, 6:3:1:1 DCM-EtOAc-MeOH-HOAc)was eluted with a 10-15-20% MeOH-DCM step gradient. The resulting paleyellow solid was washed 3× with 2.5 ml of ice-cold acetonitrile, then 2×with ether and dried in vacuo to obtain 630 mg (75%) of4-amino-3-(N-trifluoroacetyl-3-aminopropynyl)pyrazolo[3,4-d]pyrimidineβ-D-ribofuranoside (10). Identity of the product was confirmed by¹H-nmr, mass spectrometry and elemental analysis.

[0181] The nucleoside was converted to a 5′-triphosphate (11),deprotected, reacted withoxysuccinimidyl-(N-biotinoyl-6-amino)hexanoate, oroxysuccinimidyl-(N-(fluorescein-5-carboxyl)-6-amino)hexanoate, andpurified according to procedures reported elsewhere (Prober, J. M., etal., 1988, PCT 0 252 683 A2.) to give the biotin- andfluorescein-labeled nucleotides (12 a, 12 b) in >95% purity.

Example 3

[0182] Synthesis of Fluorescein- and Biotin-N6-dideoxy-pyrazalo[3,4-d]pyrimidine Nucleotides.

[0183] 1-O-acetyl-5-O(t-butyldimethylsilyl)-2,3-dideoxy-D-glycero-pentofuranose (1) and1-trimethylsilyl-4-chloropyrazolo[3,4-d]pyrimidine (13) were synthesizedaccording to literature procedures. Duelholm, K. L.; Penderson, E. B.,Synthesis 1992, 1-22; and, Robins, R. K., J. Amer Chem Soc. 1995, 78,784-790. To 2.3 g (8.3 mmol) of 1 and 1.9 g (8.3 mmol, 1 eq) of 13 in 40ml of dry DCM at 0° C. under argon was added slowly over 5 minutes 1.5mL (8.3 mmol, 1 eq) of trimethylsilyl triflate. After 30 min. 4.2 ml(41.5 mmol, 5 eq) of 1,2-diaminobutane was added rapidly and thereaction was stirred at room temperature for 1 hr. The solvent wasevaporated; the residue was dissolved in 50 ml of ethylacetate andwashed with 50 ml of saturated aqueous. NaHCO₃ and dried over Na₂SO₄,filtered and the solvent evaporated to yield 4.2 g of a yellow foam. Thefoam was dissolved in 100 ml of diethyl ether and 100 ml of hexanes wasadded to precipitate the product as an oil. The solvent was decanted andthe oil was dried under high vacuum to give 3.4 g of 15 as a pale yellowfoam. HPLC, UV and MS data were consistent with a 2:1 mixture of the α-and β-anomers.

[0184] To the crude mixture of isomers (3.4 g, 8.1 mmol, ˜50% pure) in140 ml of dry THF at 0° C. under argon was added slowly 1.0 ml of1-trifluoroacetylimidazole (8.9 mmol, 1.1 eq). The reaction was followedby RP-HPLC. An additional 5% of the acylating agent was added tocompletely convert the starting material to mixture of TFA-protectedanomers. Bergerson, R. G.; McManis, J. S J. Org. Chem 1998, 53,3108-3111. The reaction was warmed to room temperature, and then thesolvent was evaporated to about 25 ml volume and diluted with 100 ml ofethylacetate. The solution was extracted twice with 25 ml of 1% aq.NaHCO₃, once with brine, then dried over Na₂SO₄ and evaporated to afford3.4 g of yellow foam. The crude material was purified by flashchromatography on silica gel in EtOAc-hexanes to give 1.3 g of theα-anomer and 0.7 g of the β-anomer of 16 (50% total yield). The 1H-NMRand MS data were consistent with the assigned structure andstereochemistry.

[0185] To 1.3 g (2.5 mmol) of 16 (α-anomer) in 50 ml of dry THF underargon was added 1 ml (13.6 mmol) of triethylamine and 6.1 ml (37.5 mmol,15 eq) of triethylamine trihydrofluoride. After stirring for 16 hr., thesolvent was evaporated, and residual triethylamine trihydrofluorideremoved under high vacuum. Pirrung, M. C.; et al. Biorg. Med. Chem.Lett. 1994, 4, 1345-1346. The residue was dissolved in 100 ml ofethylacetate and washed carefully with 4× 100 ml of sat. aq. NaHCO₃,once with brine, then dried over Na₂SO₄ and evaporated to give 850 mg(95%) of white foam. 1H-NMR, UV and MS data were consistent with theassigned structure of the desilylated nucleoside, which was used in thenext step without further purification.

[0186] The nucleoside was converted to the triphosphate using theEckstein phosphorylation procedure (Ludwig, J. L. ; Eckstein, F. J. Org.Chem. 1989, 54, 631-635) followed by HPLC purification on a ResourceQanion exchange column (buffer A is 20 mM Tri pH8, 20% CH₃CN and buffer Bis 20 mM Tris pH8, 1 M NaCl, 20% CH3CN). ³¹P-NMR, UV and MS data wereconsistent with the structure of the triphosphate. Thetrifluoroacetyl-protecting group was removed by treatment with excessNH₄OH at 55° C. for 1 hr. followed by evaporation to dryness. The massspectral data were consistent with the aminobutyl nucleotide 17. Withoutfurther purification, the nucleotide was treated with either Biotin-NHSesters or 5-Carboxyfluorescein-NHS as described elsewhere (Prober, J.M., et al., 1988, PCT 0 252 683 A2) to form the labeled nucleotides 18a-18 d, respectively, which were purified by HPLC as described (Prober,J. M., et al., 1988, PCT 0 252 683 A2) except that, in the case of 18 a,the buffer was 20 mM sodium phosphate pH6. The ³¹P-NMR and UV data wereconsistent with the structure of the labeled analogs.

Example 4

[0187] Synthesis of N4-labeled 1,2,4-triazine-3-one β-D-ribofuranosidetriphosphates.

[0188] To a solution of 1,2,4-triazole (6.7 g; 97 mmole) in 30 mL dryACN was added POCL₃ (2.1 mL; 22 mmole) slowly with stirring under argon.After 30 minutes, the solution was cooled to 0° C., and a solution oftriethylamine (21 mL; 150 mmole) and 2′,3′,5′-tri—O-acetyl-6-azauridine(19, 4.14 g; 11 mmole (commercially available from Aldrich ChemicalCompany)) in 10 mL ACN was added. After stirring for an additional hourat room temperature, the resulting solution of activated nucleoside wastransferred dropwise to a stirring solution of 1,4-diaminobutane (46 g;524 mmole) in 20 mL MeOH. The solvents were removed in vacuo, and theresidue was taken up in water, neutralized with acetic acid, andevaporated again to dryness. The crude residue was purified bychromatography on silica gel (95:5 MeOH—NH₄OH), followed by preparativereverse-phase HPLC to yield 150 mg (0.45 mmole; 3%) of the aminobutylnucleoside (21). This was converted directly to the TFA-protectednucleoside (22) by reaction with 1-trifluoroacetylimidazole (300 uL; 1.8mmole) in 3 ml ACN at 0° C. for 2 hours, evaporating the solvent andpurifying by flash chromatography (1:9 MeOH-DCM). Yield 175 mg (0.42mmole; 93%). Identity of the product was confirmed by ¹H-nmr and massspectrometry.

[0189] The nucleoside was converted to a 5′-triphosphate, deprotected,reacted with oxysuccinimidyl-(N-biotinoyl-6-amino)hexanoate, oroxysuccinimidyl-(N-(fluorescein-5-carboxyl)-6-amino)hexanoate, andpurified according to procedures reported elsewhere (Prober, J. M., etal., 1988, PCT 0 252 683 A2.) to give the biotin- andfluorescein-labeled nucleotides (23 a, 23 b) in >95% purity.

Example 5

[0190] Synthesis of Biotin and Fluorescein C5-Labeled1,2,4-Triazine-3,5-dione Riboside Triphosphates.

[0191] 5-Formyl-6-azauracil (24) is prepared according to literatureprocedures. See, Scopes, D.I.C. 1986, J. Chem. Med., 29, 809-816, andreferences cited therein. Compound 24 is reacted with the phosphoniumylide of 25, which is formed by treating 25 with catalytic t-butoxide,to provide the phthalimidoyl-protected allylamine 26. Protectedallylamine 26 is ribosylated to provide β-anomer 28 upon reaction of 26with β-D-pentofuranoside 27 (commercially available from Aldrich)according to the procedure of Scopes et al. 1986, J. Chem. Med., 29,809-816. β-ribonucleoside 28 is deprotected with anhydrous hydrazine inTHF to provide allylamine 29. Reaction of primary amine 29 withtrifluoroacetylimidazole in THF affords the protected amine 30.

[0192] Nucleoside 30 is converted to a 5′-triphosphate, deprotected,reacted with oxysuccinimidyl-(N-biotinoyl-6-amino)hexanoate oroxysuccinimidyl-(N-(fluorescein-5-carboxy)-6-amino)hexanoate andpurified according to procedures reported elsewhere (Prober, J. M., etal. 1988, PCT 0 252 683 A2), giving, respectively, the biotin- andfluorescein-labeled nucleotides 31 a and 31 b.

Example 6

[0193] Synthesis of Biotin and Fluorescein C5-Labeled5-Amino-1,2,4-triazine-3-one Riboside Triphosphates.

[0194] β-ribonucleoside 28, described above, is treated with SOCl₂ orPOCl₃ and subsequently reacted with ammonia to provide the4-amino-1,3,6-triazine nucleoside 32. The phthalimide group of 32 isremoved upon reaction with hydrazine, and the resulting primary amine isprotected to afford nucleoside 33. Nucleoside 33 is converted to a5′-triphosphate, deprotected, reacted withoxysuccinimidyl-(N-biotinoyl-6-amino)hexanoate oroxysuccinimidyl-(N-(fluorescein-5-carboxy)-6-amino)hexanoate andpurified according to procedures reported elsewhere (Prober, J. M., etal. 1988, PCT 0 252 683 A2), giving, respectively, the biotin- andfluorescein-labeled nucleotides 34 a and 34 b.

Example 7

[0195] Procedure for HPLC Analysis of Enzymatic Incorporation ofModified Nucleotides.

[0196] Reaction Conditions

[0197] TdT

[0198] 3 uM dT_(16 template)

[0199] 15(30) uM NTP

[0200] 40 U TdT (Promega)

[0201] 1× buffer, pH 7.5 (Promega)

[0202] Procedure: incubate 1 hr. at 37° C., then for 10 min. at 70° C.,followed by the addition of EDTA (2 mM final concentration) in a volumeof 50 uL

[0203] HPLC Analysis

[0204] Materials and Reagents

[0205] 4.6 mm×250 mm Nucleopac PA-100 ion-exchange column (Dionex)buffer A: 20 mM NaOH (or 20 mM Tris pH 8, in the case of TdTincorporation of nucleotide triphoshates that are not dye-labeled)buffer B: 20 mM NaOH, 1M NaCl (or 20 mM Tris pH 8, 1M NaCI, in the caseof TdT incorporation of nucleotide triphoshates that are notdye-labeled)

[0206] General Procedure

[0207] Dilute the reaction with 50 uL of buffer A. Inject 50 uL of thissample onto the HPLC column and fractionate using a gradient of 5 to100% buffer B over 30 minutes at a flow rate of 1 mL/min. Detect thepeaks simultaneously at 260 nm absorbance and the absorbance maximum ofthe dye (or the fluorescence emission maximum of the dye).

[0208] The incorporation efficiency is expressed as the fraction ofoligonucleotide that is labeled. This number is determined by dividingthe peak area measured at 260 nm absorbance of the labeledoligonucleotide by the sum of the peak areas of the unlabeled andlabeled oligonucleotide. (The retention time of fluorescein-labeled dT₁₆is on the order of 2 to 3 min. longer than the unlabeled dT_(16.)) Theerror in this type of assay is about 10%. The percentage labelingefficiency for 4 types of nucleic acid labeling compounds is shown belowin Table 1. TABLE 1

% Labelling Efficiency [TdT] = R= B= 40 U 160 U H

X = -BIOTIN (18a) —CO(CH₂)₅NH-BIOTIN (18b) —CO(CH₂)₅NHCO(CH₂)₅NH-BIOTIN(18c) 5-carboxyFluorescein (18d) 48 41 57 60 100  96 94 98 OH

X = -BIOTIN (12a) 5-carboxyFluorescein (12b) 25 53 84 97 H

—CO(CH₂)₅NH-BIOTIN (8a) 5-carboxyFluorescein (8b) 88 94 94 97 OH

X = -BIOTIN (23a) 5-carboxyFluorescein (23b) 47 67 85 98

Example 8

[0209] Hybridization Studies Of Labeled Imidazole Carboxamide (“ITP”)and 4-Aminopyrazolo[3,4-d]pyrimidine (“APPTP) Nucleotides.

[0210] The performance of the labeled imidazolecarboxamide and4-aminopyrazolo[3,4-d]pyrimidine nucleotides was evaluated in a p53assay using standard GeneChip® product protocols (Affymetrix, Inc.,Santa Clara, Calif.), which are described, for example, in detail in theGeneChip® p53 assay package insert. The sample DNA used in theseexperiments was the plasmid “p53mut248.” The labeled nucleotide analogwas substituted for the usual labeling reagent (Fluorescein-N6-ddATP orBiotin-M-N6-ddATP (wherein M=aminocaproyl), from NEN, part #'s NEL-503and NEL-508, respectively). Labeling reactions were carried out usingboth the standard amount of TdT enzyme specified in the assay protocol(25 U) and with 100 U of enzyme. After labeling, Fluorescein-labeledtargets were hybridized to the arrays and scanned directly. Inexperiments using the biotin-labeled targets, the GeneChip® chips werestained in a post-hybridization step with a phycoerythrin-streptavidinconjugate (PE-SA), prior to scanning, according to described procedures(Science 280:1077-1082 (1998)).

[0211]FIG. 9 shows comparisons of the observed hybridizationfluorescence intensities for the 1300 bases called in the “Unit-2” partof the chip. In the lower plot, intensities for the Fluorescein-ddITP (8b) labeled targets are plotted against those for the standardFluorescein-N6-ddATP labeled targets (control), both at 25 U of TdT. Theobserved slope of ˜0.75 indicates that the labeling efficiency of 8 bwas about 75% of that of Fluorescein-N6-ddATP under these conditions. Inthe upper plot, the same comparison is made, except that 100 U of TdTwas used in the 8 b labeling reaction. The slope of ˜1.1 indicatesequivalent or slightly better labeling than the standardFluorescein-N6-ddATP/25 U control reaction.

[0212]FIG. 10 shows comparisons of the observed hybridizationfluorescence intensities for the 1300 bases called in the “Unit-2” partof the chip. Intensities for the Biotin-(M)₂-ddAPPTP (18 c,M=aminocaproyl linker; referred to as Biotin-N4-ddAPPTP in FIG. 10)labeled targets (after PE-SA staining) are plotted against those for thestandard Biotin-M-N6-ddATP labeled targets (control), both at 25 U ofTdT. The observed slope of ˜0.3 indicates that the labeling efficiencywithBiotin-(M)₂-ddAPPTP (18 c) was about 30% of that ofBiotin-M-N6-ddATP under these conditions.

[0213]FIG. 11 shows comparisons of the observed hybridizationfluorescence intensities for the 1300 bases called in the “Unit-2” partof the chip. In the lower plot, intensities for the Biotin-M-ddITP (8 a,M=aminocaproyl; referred to as Bio-ddITP in FIG. 11) labeled targets areplotted against those for the standard Biotin-M-N6-ddATP labeled controltargets, both at 25 U of TdT. The observed slope of ˜0.4 indicates thatthe labeling efficiency with 8 a was about 40% of that ofBiotin-M-N6-ddATP under these conditions. In the upper plot, the samecomparison is made, except that 100 U of TdT was used in the 8 alabeling reaction. The slope of ˜1.1 indicates equivalent or slightlybetter labeling than the standard Biotin-M-N6-ddATP/25 U controlreaction.

[0214]FIG. 12 shows a comparison of the overall re-sequencing(base-calling) accuracy, for both strands, obtained usingFluorescein-ddITP labeled targets at both 25 U and 100 U of TdT, as wellas the standard Fluorescein-N6-ddATP/25 U TdT labeled “control” targets.FIG. 13 shows a similar comparison for the targets labeled withbiotin-M-ddITP (8 a; referred to as Biotin-ddITP in FIG. 13) andbiotin-M-N6-ddATP “control,” followed by PE-SA staining. FIG. 14 shows acomparison of re-sequencing accuracy using Biotin-(M)₂-ddAPPTP/100 U TdTand Biotin-M-N6-ddATP/25 U TdT. These data indicate that labeledimidazolecarboxamide and 4-aminopyrazolo[3,4-d]pyrimidinedideoxynucleotide analogs can be used for DNA target labeling inhybridization-based assays and give equivalent performance to thestandard labeled-N6-ddATP reagent.

Example 9

[0215] The performance of the biotin-labeled imidazolecarboxamide and4-aminopyrazolo[3,4-d]pyrimidine nucleotides (“biotin-M-ITP” (8 a) and “biotin-(M)₂-APPTP” (18 c)) was evaluated using a single-nucleotidepolymorphism genotyping GeneChip® chip array. Published protocols (D. G.Wang, et al., 1998, Science 280: 1077-82.) were used in theseexperiments, except for the following variations: 1) labeling reactionswere carried out using both the standard amount of TdT enzyme specifiedin the published protocol (15 U), or three-fold (45 U) enzyme; 2)substitution of the labeled nucleotide analog for the standard labelingreagent (Biotin-N6-ddATP, from NEN: P/N NEL-508); 3) the labelednucleotide analog was used at either twice the standard concentrationspecified in the published protocol (25 uM), or at six-fold (75 uM).After labeling, biotin-labeled targets were hybridized to the arrays,stained with a phycoerythrin-streptavidin conjugate (PE-SA), and thearray was scanned and analyzed according to the published procedure.

[0216] The data is shown in the Table 2 below. As indicated by the meanintensities of the observed hybridization signal (averaged over theentire array), labeling efficiency with biotin-M-ITP (8 a) at 25 uM wasas good as Biotin-N6-ddATPat 12.5 uM, and even higher intensity wasgained by using 8 a at 75 uM (entries 1-3; 7,8). Compared with thecontrol, this analog provided equivalent or better assay performance,expressed as the ratio of correct base calls. Somewhat lower mean signalintensities are observed with biotin-(M)₂-APPTP (18 c), reflecting thelower incorporation efficiency of this analog, but equivalent assayperformance could still be achieved with this analog, using somewhathigher enzyme and nucleotide concentrations (entries 3-6). TABLE 2Comparison of Polymorphism Chip Data Correct Base Units Mean Call EntrySample Nucleotide [Nucleotide] TdT Intensity Ratio 1 A Biotin-M- 75 15164 0.98 ddIcTP (8a) 2 A Biotin-M- 75 45 235 0.98 ddIcTP (8a) 3 BBiotin-N6- 12.5 15 138 0.95 con- M-ddATP trol (NEL 508) 4 B Biotin-N4-25 15 37 0.88 (M)₂- ddAppTP (18c) 5 B Biotin-N4- 75 15 35 0.92 (M)₂-ddAppTP (18c) 6 B Biotin-N4- 75 45 87 0.95 (M)₂- ddAppTP (18c) 7 BBiotin-M- 25 15 116 0.95 ddIcTP (8a) 8 B Biotin-M- 75 15 149 0.95 ddIcTP(8a)

1. A nucleic acid labeling compound of the following structure:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₁, SR₁ or NHR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, For OR₁, wherein R₁ is H, alkyl or aryl; L is amido alkyl; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about
 3. 2. A nucleic acid labeling compound accordingto claim 1 , wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, whereinR₁ is H, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl oraryl; L is —C(O)NH(CH₂)_(n)NH—, wherein n is an integer ranging fromabout 2 to about 10; Q is biotin or a carboxyfluorescein; and, M is—CO(CH₂)₅NH—, wherein m is 1 or
 0. 3. A nucleic acid labeling compoundaccording to claim 2 , wherein Y is H or OH; Z is H or OH; L is—C(O)NH(CH₂)₄NH—; Q is biotin; and, M is —CO(CH₂)₅NH, wherein m is
 1. 4.A nucleic acid labeling compound according to claim 2 , wherein Y is Hor OH; Z is H or OH; L is —C(O)NH(CH₂)₄NH—; Q is 5-carboxyfluorescein;and m is
 0. 5. A nucleic acid labeling compound of the followingstructure:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₁, SR₁ or NHR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, For OR₁, wherein R₁ is H, alkyl or aryl; L is amino alkyl; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about
 3. 6. A nucleic acid labeling compound accordingto claim 5 , wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁, whereinR₁ is H, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H, alkyl oraryl; L is —NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2to about 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH—or —CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or
 0. 7. A nucleic acidlabeling compound according to claim 6 , wherein Y is H or OH; Z is H orOH; L is —NH(CH₂)₄NH—; Q is biotin; and, m is
 0. 8. A nucleic acidlabeling compound according to claim 6 , wherein Y is H or OH; Z is H orOH; L is —NH(CH₂)₄NH—; Q is 5-carboxyfluorescein; and, m is
 0. 9. Anucleic acid labeling compound of the following structure:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₁, SR₁ or NHR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, For OR₁, wherein R₁ is H, alkyl or aryl; L is alkynyl alkyl; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about
 3. 10. A nucleic acid labeling compoundaccording to claim 9 , wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₁,wherein R₁ is H, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ is H,alkyl or aryl; L is —C≡C(CH₂)_(n)NH—, wherein n is an integer rangingfrom about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is—CO(CH₂)₅NH—, wherein m is 1 or
 0. 11. A nucleic acid labeling compoundaccording to claim 10 , wherein Y is H or OH; Z is H or OH; L is—C≡CCH₂NH—; Q is biotin; and, m is
 1. 12. A nucleic acid labelingcompound according to claim 10 , wherein Y is H or OH; Z is H or OH; Lis —C≡CCH₂NH—; Q is 5-carboxyfluorescein; and, m is
 1. 13. A nucleicacid labeling compound of the following structure:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₁, SR₁ or NHR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, For OR₁, wherein R₁ is H, alkyl or aryl; L is amino alkyl; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about
 3. 14. A nucleic acid labeling compoundaccording to claim 13 , wherein A is H or H₄O₉P₃—; X is O; Y is H orOR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, F or OR₁, wherein R₁ isH, alkyl or aryl; L is —NH(CH₂)_(n)NH—, wherein n is an integer rangingfrom about 2 to about 10; Q is biotin or carboxyfluorescein; and, M is—CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or
 0. 15. Anucleic acid labeling compound according to claim 14 , wherein Y is H orOH; Z is H or OH; L is —NH(CH₂)₄NH—; Q is biotin; and, M is—CO(CH₂)₅NH—, wherein m is
 1. 16. A nucleic acid labeling compoundaccording to claim 14 , wherein Y is H or OH; Z is H or OH; L is—NH(CH₂)₄NH—; Q is 5-carboxyfluorescein; and, M is —CO(CH₂)₅NH—, whereinm is
 1. 17. A nucleic acid labeling compound of the following structure:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₁, SR₁ or NHR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, For OR₁, wherein R₁ is H, alkyl or aryl; L is functionalized alkyl,alkenyl alkyl or alkynyl alkyl; Q is a detectable moiety; and, M is aconnecting group, wherein m is an integer ranging from 0 to about
 3. 18.A nucleic acid labeling compound according to claim 17 , wherein A is Hor H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ is H, alkyl or aryl; Z isH, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl; L is—CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or
 0. 19. A nucleic acid labelingcompound according to claim 18 , wherein Y is H or OH; Z is H or OH; Lis —CH═CHCH₂NH—; Q is biotin; and, m is
 0. 20. A nucleic acid labelingcompound according to claim 18 , wherein Y is H or OH; Z is H or OH; Lis —CH═CHCH₂NH—; Q is 5-carboxyfluorescein; and, m is
 0. 21. A nucleicacid labeling compound of the following structure:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₁, SR₁ or NHR₁, wherein R₁ is H, alkyl or aryl; Z is H, N₃, For OR₁, wherein R₁ is H, alkyl or aryl; L is functionalized alkyl,alkenyl alkyl or alkynyl alkyl; Q is a detectable moiety; and, M is aconnecting group, wherein m is an integer ranging from 0 to about
 3. 22.A nucleic acid labeling compound according to claim 21 , wherein A is Hor H₄O₉P₃—; X is O; Y is H or OR₁, wherein R₁ is H, alkyl or aryl; Z isH, N₃, F or OR₁, wherein R₁ is H, alkyl or aryl; L is—CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or
 0. 23. A nucleic acid labelingcompound according to claim 22 , wherein Y is H or OH; Z is H or OH; Lis —CH═CHCH₂NH—; Q is biotin; and, m is
 0. 24. A nucleic acid labelingcompound according to claim 22 , wherein Y is H or OH; Z is H or OH; Lis —CH═CHCH₂NH—; Q is 5-carboxyfluorescein; and, m is
 0. 25. A nucleicacid derivative produced by coupling a nucleic acid labeling compoundaccording to claim 2 with a nucleic acid.
 26. A nucleic acid derivativeproduced by coupling a nucleic acid labeling compound according to claim6 with a nucleic acid.
 27. A nucleic acid derivative produced bycoupling a nucleic acid labeling compound according to claim 10 with anucleic acid.
 28. A nucleic acid derivative produced by coupling anucleic acid labeling compound according to claim 14 with a nucleicacid.
 29. A nucleic acid derivative produced by coupling a nucleic acidlabeling compound according to claim 18 with a nucleic acid.
 30. Anucleic acid derivative produced by coupling a nucleic acid labelingcompound according to claim 22 with a nucleic acid.
 31. A hybridizationproduct, wherein the hybridization product comprises the nucleic acidderivative according to claim 25 bound to a complementary probe.
 32. Thehybridization product according to claim 31 , wherein the probe isattached to a glass chip.
 33. A hybridization product, wherein thehybridization product comprises the nucleic acid derivative according toclaim 26 bound to a complementary probe.
 34. The hybridization productaccording to claim 33 , wherein the probe is attached to a glass chip.35. A hybridization product, wherein the hybridization product comprisesthe nucleic acid derivative according to claim 27 bound to acomplementary probe.
 36. The hybridization product according to claim 35, wherein the probe is attached to a glass chip.
 37. A hybridizationproduct, wherein the hybridization product comprises the nucleic acidderivative according to claim 28 bound to a complementary probe.
 38. Thehybridization product according to claim 37 , wherein the probe isattached to a glass chip.
 39. A hybridization product, wherein thehybridization product comprises the nucleic acid derivative according toclaim 29 bound to a complementary probe.
 40. The hybridization productaccording to claim 39 , wherein the probe is attached to a glass chip.41. A hybridization product, wherein the hybridization product comprisesthe nucleic acid derivative according to claim 30 bound to acomplementary probe.
 42. The hybridization product according to claim 41, wherein the probe is attached to a glass chip.
 43. A method ofsynthesizing a labeled nucleic acid comprising attaching a nucleic acidlabeling compound according to claim 2 to a nucleic acid.
 44. A methodof synthesizing a labeled nucleic acid comprising attaching a nucleicacid labeling compound according to claim 6 to a nucleic acid.
 45. Amethod of synthesizing a labeled nucleic acid comprising attaching anucleic acid labeling compound according to claim 10 to a nucleic acid.46. A method of synthesizing a labeled nucleic acid comprising attachinga nucleic acid labeling compound according to claim 14 to a nucleicacid.
 47. A method of synthesizing a labeled nucleic acid comprisingattaching a nucleic acid labeling compound according to claim 18 to anucleic acid.
 48. A method of synthesizing a labeled nucleic acidcomprising attaching a nucleic acid labeling compound according to claim22 to a nucleic acid.
 49. A method of detecting a nucleic acidcomprising incubating a nucleic acid derivative according to claim 25with a probe.
 50. A method according to claim 49 , wherein the probe isattached to a glass chip.
 51. A method of detecting a nucleic acidcomprising incubating a nucleic acid derivative according to claim 26with a probe.
 52. A method according to claim 51 , wherein the probe isattached to a glass chip.
 53. A method of detecting a nucleic acidcomprising incubating a nucleic acid derivative according to claim 27with a probe.
 54. A method according to claim 53 , wherein the probe isattached to a glass chip.
 55. A method of detecting a nucleic acidcomprising incubating a nucleic acid derivative according to claim 28with a probe.
 56. A method according to claim 55 , wherein the probe isattached to a glass chip.
 57. A method of detecting a nucleic acidcomprising incubating a nucleic acid derivative according to claim 29with a probe.
 58. A method according to claim 57 , wherein the probe isattached to a glass chip.
 59. A method of detecting a nucleic acidcomprising incubating a nucleic acid derivative according to claim 30with a probe.
 60. A method according to claim 59 , wherein the probe isattached to a glass chip.